Members of the glucose transporter family (GLUT, SLC2) are involved in diabetes, obesity, cancer, and other diseases. There are 14 human GLUTs, which vary in tissue distribution and substrate affinity and specificity. Their exploitation as therapeutic targets has lagged due to lack of specific inhibitors and inadequate understanding of the molecular basis for their transport differences. Crystal structures of several GLUTs and their homologues are quite similar and reveal two major transporter conformations, consistent with an alternating mechanism of transport. Yet, the molecular basis of substrate affinity and specificity is unclear. Also, there is a critical need for GLUT-specific ligands that could be further optimized into drugs, diagnostic markers and investigative tools, to probe the role of particular GLUTs in disease. Our long-term goal is to determine how modulation of carbohydrate transport is involved in health and disease. The overall objective in this application is to identify new GLUT-specific ligands and the molecular basis of ligand specificity for six GLUTs: GLUT1-5 and 9. Our central hypothesis is that ligand specificity and affinity in GLUTs depends on the conformation of the transporter and is mediated by both substrate binding site residues and long-range interactions involving the soluble loops. Our preliminary data on GLUT5 and GLUT homologues Hxt, Gal2 and GlcPSe, support this hypothesis. The rationale for the proposed research is that identification of GLUT-selective ligands and GLUT-specific molecular interactions responsible for ligand recognition and affinity will enable development of specific modulators for GLUTs, and ultimately their pharmacological control. We will test our hypothesis with the following two specific aims: 1) identify conformation-specific ligands for GLUT1-5 and GLUT9; and 2) determine key residues and regions responsible for ligand specificity and affinity in GLUT1-5 and GLUT9. For the first aim, we will combine in silico screening of small molecule libraries against inward- and outward-facing conformations of GLUT1-5 and 9 models with in vitro validation of top ranked candidates, for inhibition and selectivity, in two different GLUT-individualized transport systems. We established the feasibility of this approach by identifying the first potent and specific inhibitor for GLUT5. For the second aim the transport activity of mutants in GLUT-specific ligand binding sites (ligands known or determined from Aim 1) and of GLUT chimeras constructed by swapping soluble loops between selected pairs of GLUTs, will be examined for changes in substrate and inhibitor specificity and/or affinity. Ligand binding sites will be determined through computational ligand docking and crystal structure determination of liganded GLUT complexes. The approach is innovative because it addresses ligand specificity in GLUT family comprehensively by assembling an integrated combination of methods. The proposed research is significant, because it is expected to lead to discovery of novel inhibitors and alternate substrates for GLUT1-5 and 9, and illuminate the molecular determinants of the functional variability within the GLUT family.